The present invention relates to a process for preparing optically active chloropropanediol derivative which comprises treating an inexpensive (RS)-chloropropanediol derivative with a nitroxyl compound in the presence of an oxidant to convert to an chlorohydroxyacetone derivative, and stereospecifically reducing the chlorohydroxyacetone derivative under an enzyme source having an activity which allow an chlorohydroxy acetone derivative to be reduced, and an important intermediate thereof. An optically active chloropropanediol derivative, especially (S)-3-benzyloxy-1-chloro-2-propanol is useful compound as an intermediate of a medicine.
As the process for preparing chlorohydroxyacetone derivative, for example, the process for oxidizing 1-benzoyloxy-3-chloro-2-propanol with DCC (Journal of Medicinal Chemistry, (20), 5, 1997) is known. However, such process has problems of using harmful heavy metals and of low yield.
As the reduction process of chlorohydroxyacetone with, for example, a microorganism, the following processes are known. (1) A process for stereoselective reduction of 1-acetyloxy-3-chloro-2-propanone with an microorganism (JP-A-11-103878), and (2) a process for R selective reduction of 1-chloro-3-(p-acetoamidephenoxy)-2-propanone with a microorganism (JP-A-02-295970). However, these processes have a problem in stereoselectivity, productivity or the like.
As mentioned above, these processes have a problem to be improved as an industrial preparation process.
As a result of intense studies for the purpose of preparing optically active chloropropanediol derivatives, especially (S)-1-benzyloxy-3-chloro-2-propanol effectively in a high optical purity, a process for preparing an optically active chloropropanediol derivative, which comprises treating inexpensive (RS)-chloropropanediol derivative with a nitroxyl compound in the presence of an oxidant to convert to an chlorohydroxyacetone derivative, and stereospecifically reducing the chlorohydroxyacetone derivative under an enzyme source having an activity which allow an chlorohydroxy acetone derivative to be reduced, and 1-benzyloxy-3-chloro-2-propanon which is one of important intermediates thereof have been found to complete the present invention.
That is to say, the present invention relates to a chlorohydroxyacetone derivative represented by the formula (1); 
wherein R1 is an aralkyl group which may be substituted with a group having 1 to 15 carbon atoms.
In the above-mentioned chlorohydroxyacetone derivative, R1 is preferably benzyl group or a substituted benzyl group.
The present invention also relates to a process for preparing a chlorohydroxyacetone derivative represented by formula (3); 
wherein R2 is an alkyl group having 1 to 10 carbon atoms, an aryl group which may be substituted with a group having 1 to 15 carbon atoms, an aralkyl group which may be substituted with a group having 1 to 15 carbon atoms, an alkylsulfonyl group having 1 to 10 carbon atoms, an arylsulfonyl group which may be substituted with a group having 1 to 15 carbon atoms, an alkylcarbonyl group having 1 to 10 carbon atoms or an arylcarbonyl group which may be substituted with a group having 1 to 15 carbon atoms,
which comprises allowing a chloropropanediol derivative represented by the formula (2); 
wherein R2 is the same as the defined above, to react with a nitroxyl compound represented by the formula (7); 
wherein each of R4, R5, R6 and R7 is an alkyl group which may be the same or different, R8 is hydrogen atom or electron-releasing group, in the presence of an oxidizing agent.
Furthermore, the present invention relates to a process for preparing an optically active chloropropanediol derivative represented by the formula (4); 
wherein R2 is the same as the mentioned above, which comprises allowing chlorohydroxy acetone derivative represented by the formula (3) obtained by the above-mentioned preparing process to be stereospecifically reduced in the presence of an enzyme source having an activity to reduce it stereospecifically.
Additionally, the present invention relates to a process for preparing an optically active chloropropanediol derivative represented by the formula (6); 
wherein R3 is an alkyl group having 1 to 10 carbon atoms, an aryl group which may be substituted with a group having 1 to 15 carbon atoms, an aralkyl group which may be substituted with a group having 1 to 15 carbon atoms, an alkylsulfonyl group having 1 to 15 carbon atoms, an arylsulfonyl group which may be substituted with a group having 1 to 15 carbon atoms, which comprises allowing a chlorohydroxyacetone derivative represented by the formula (5); 
wherein R3 is the same as the mentioned above, to be stereospecifically reduced in the presence of an enzyme source having an activity to reduce the chlorohydroxy acetone derivative represented by the above-mentioned formula (5) stereospecifically.
The present invention is explained in detail below.
In the aralkyl group which may be substituted with a group having 1 to 15 carbon atoms represented by R1 in the formula (1), the aralkyl group include benzyl group, phenylethyl group, phenylpropyl group, naphthylmethyl group. Among them, benzyl group is preferable. The substituent of the aralkyl group includes an alkyl group having 1 to 8 carbon atoms, an alkoxy group having 1 to 8 carbon atoms, an acyl group having 1 to 10 carbon atoms, an aliphatic amide group having 1 to 5 carbon atoms, an aliphatic ether having 1 to 5 carbon atoms, an unsaturated aliphatic ether having 1 to 5 carbon atoms, a halogen atom such as fluorine, chlorine, iodine or bromine, hydroxyl group, thiol group, nitro group, amino group, cyano group, an aryl group such as phenyl group or naphthyl group. Among them, the halogen atom such as fluorine, chlorine, iodine or bromine is preferable.
In the compounds represented by each of the formula (2) to (6), the alkyl group having 1 to 10 carbon atoms represented by R2 or R3 includes methyl group, ethyl group, propyl group, isopropyl group, n-butyl group, isobutyl group, sec-butyl group, t-butyl group or the like. Among them, t-butyl group is preferable. In the aryl group which may be substituted with a group having 1 to 15 carbon atoms, the aryl group includes phenyl group, naphthyl group, pyridyl group, indolinyl group or the like. Among them, phenyl group is preferable. The substituent of the aryl group includes an alkyl group having 1 to 8 carbon atoms, an alkoxy group having 1 to 8 carbon atoms, an acyl group having 1 to 10 carbon atoms, an aliphatic amide group having 1 to 5 carbon atoms, an aliphatic ether having 1 to 5 carbon atoms, an unsaturated aliphatic ether having 1 to 5 carbon atoms, a halogen atom such as fluorine, chlorine, iodine or bromine, hydroxyl group, thiol group, nitro group, amino group, cyano group, an aryl group such as phenyl group or naphthyl group. Among them, the halogen atom such as fluorine, chlorine, iodine or bromine is preferable. The number of substituents is preferably 0 to 3. As the alkylsulfonyl group having 1 to 15 carbon atoms, methane sulfonyl group is preferable. The arylsulfonyl group which may be substituted with a group having 1 to 15 carbon atoms includes phenylsulfonyl group, p-toluenesulfonyl group or p-nitruphenylsulfonyl group. Among them, p-toluenesulfonyl group is preferable.
In the compounds represented by each of the formula (2) to (4), the alkylcarbonyl group having 1 to 10 carbon atoms represented by R2 includes acetyl group, ethylcarbonyl group, propylcarbonyl group or the like. Among them, acetyl group is preferable. The arylcarbonyl group which may be substituted with a group having 1 to 15 carbon atoms includes p-bromobenzoly group or the like. Among them, benzoyl group and p-nitrobenzoly group are preferable.
It is known that, chloropropanediol derivative used as a raw material and represented by the formula (2) in the present invention, for example, (RS)-1-chloro-3-benzyloxy-2-propanol can be easily prepared by allowing epichlorohydrin to react with benzylalcohol in the presence of a Lewis acid (JP-A-02-211).
In the present invention, first chlorohydroxyaceton derivative represented by the formula (3) is prepared by oxidizing (RS)-chloropropanediol derivative represented by the formula (2) with nitroxyl compound represented by the formula (7) in the presence of an oxidant.
The oxidation process of the present invention can be carried out by adding chloropropanediol derivative and nitroxyl compound to an appropriate solvent, and by dropping and stirring oxidant thereto. The reaction condition is not limited particularly. Generally, the reaction temperature is 0 to 70xc2x0 C., preferably 0 to 20xc2x0 C. The reaction pH is 4 to 10, preferably 7 to 9. The concentration of substrate is 0.1 to 50% (w/v), preferably 1 to 50% (w/v). The amount of nitroxyl compound to be added is 0.001 to 1 equivalent to substrate, preferably 0.01 to 0.1 equivalent. As the reaction solvent, acetic acid, toluene or methylen chloride is used.
In the nitroxy compound represented by the formula (7), the substituent represented by R4 to R7 includes methyl group. As the substituent represented by R8, hydrogen atom, methoxy group, benzoyloxy group, acethylamino group, oxytho group or hydroxyl group is preferable and hydrogen atom and acethylamino group is more preferable. As concrete examples of the nitroxyl compound, 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO), 4-acethylamino-2,2,6,6-tetramethylpiperidine-1-oxyl, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl, 4-benzoyloxy-2,2,6,6-tetramethylpiperidine-1-oxyl, 4-methoxy-2,2,6,6-tetramethylpiperidine-1-oxyl and 4-oxtho-2,2,6,6-tetramethylpiperidine-1-oxyl. Among them, 2,2,6,6-tetramethylpiperidine-1-oxyl or 4-acethylamino-2,2,6,6-tetramethylpiperidine-1-oxyl is preferable.
Examples of the oxidantinclude sodium hypochlorite, bleaching powder or chlorine. Among them, sodium hypochlorite is preferable.
Chlorohydroxyaceton derivative obtained by the oxdation reaction can be purified according to general procedure. For example, 1-benzyloxy-3-chloro-2-propanon can be purified by extraction with an organic solvent such as ethyl acetate or toluene from the mixture, removing the solvent under reduced pressure, treating the same by carrying out distillation under reduced pressure or chromatography. Also, it can be employed in a subsequent step without such purification.
By the way, among the prepared chlorohydroxyacetone dervatives, the compound whose R2 is benzyl group, that is 1-benzyloxy-3-chloro-2-propanon is a novel compound whose usefulness is found by the present inventor.
In the present invention, an optically active chloropropanediol derivative can be prepared by reducing the thus-obtained chlorohydroxyacetone derivative in the presence of enzyme source having an activity to reduce the same stereoselectively.
In the reduction process of the present invention, example of the enzyme source having an activity to reduce chlorohydroxyacetone derivative stereospecifically includes the enzyme source derived from microorganism belonging to Candida genus, Cryptococcus genus, Debaryomyces genus, Dekkera genus, Dipodascus genus, Galacctomyces genus, Geotrichum genus, Hansenula genus, Komagataella genus, Kluyveromyces genus, Pichia genus, Rhodotorula genus, Yarrowia genus, Sporidiobolus genus, Yamadazyma genus, Torulaspora genus, Torulopsis genus, Trichosporon genus, Alcaligenes genus, Rhodococcus genus, Micrococcus genus, Absidia genus, Acrotheca genus, Aspergillus genus, Chaetomidium genus, Gibberella genus, Mortierella genus or Eupenicillium genus.
Concretely, preferable example includes the enzyme source derived from a microorganism selected from the group consisting of Candida galacta species, Candida arborea species, Candida catenulata species, Candida etchellsii species, Candida glabrata species, Candida gropengiesseri species, Candida guilliermondii species, Candida magnoliae species, Candida maltosa species, Candida parapsilosis species, Candida pararugosa species, Candida pinus species, Candida rugosa species, Candida sake species, Candida saitoana species, Candida sorbophila species, Candida tropicalis species, Cryptococcus humicolus species, Cryptococcus laurentii species, Cryptococcus terreus species, Debaryomyces hansenii species, Debaryomyces sp. H list et Guiel species, Debaryomyces robertsiae species, Debaryomyces castellii species, Debaryomyces psedopolymorphus species, Dekkera anomala species, Dipodascus armillariae species, Dipodascus ovetensis species, Galacctomyces reessii species, Geotrichum fermentans species, Geotrichum fragrans species, Hansenula polymorpha DL1 species, Komagataella pastoris species, Kluyveromyces thermotolerans species, Pichia membranaefaciens species, Pichia naganishii species, Rhodotorula glutinis species, Yarrowia lipolytica species, Sporidiobolus johnsonii species, Yamadazyma farinosa species, Torulaspora globosa species, Trichosporon aquatile species, Alcaligenes xylosaxidens subsp. denitrificans species, Rhodococcus erythropolis species, Rhodococcus equi species, Micrococcus luteus species, Absidia glauca species, Acrotheca cerophila species, Aspergillus japonicus species, Aspergillus parasiticus species, Aspergillus terreus species, Chaetomidium fimeti species, Gibberella fujikuroi species, Mortierella ramanniana var. angulispora species and Eupenicillium baarnense species. More preferable example includes the enzyme source derived from Candida magnoliae IFO 0705.
The above-mentioned microorganism being the enzyme source may be either wild strain or variant. As the enzyme source, a microorganism which is derived by genetic procedure such as cell fusion or genetic manipulation can be used.
The microorganism producing the present enzyme can be obtained according to the method which comprises a step of isolating and/or purifying those enzymes to determine the complete or partial amino acid sequence thereof, a step of obtaining the DNA sequence encoding the enzyme according to the amino acid sequence, a step of introducing the DNA into other microorganisms to obtain a transformed microorganism, and a step of cultivating the transformed microorganism to obtain the present enzyme (WO 98/35025). Examples thereof include a transformed cell transformed by plasmid comprising DNA encoding the above carbonyl-reductase and DNA encoding an enzyme capable of regenerating coenzyme to which the enzyme depends. Preferably, examples are a transformed cell such that the enzyme capable of regenerating coenzyme is glucose dehydrogenase, a transformed cell such that the glucose dehydrogenase is derived from Bacillus megaterium, a transformed cell such that the plasmid is pNTS1G, a transformed cell such that the transformed cell is Escherichia coli and the like. Concretely, E. coli HB101 (pNTS1G) accession No. FERM-BP 5835 is more preferable.
The medium for the microorganism is not particularly limited as long as the microorganism is proliferated thereon. Examples thereof are a normal liquid medium containing sugar such as glucose or sucrose; alcohol such as ethanol or glycerol; fatty acid such as oleic acid or stearic acid and ester thereof; and oil such as rapeseed oil or soybean oil as carbon source, ammonium sulfate, sodium nitrate, peptone, Casamino acid, cone steep liquor, bran, yeast extract or the like as nitrogen source, magnesium sulfate, sodium chloride, calcium carbonate, dipotassium hydrogenphosphate, potassium dihydrogenphosphate as inorganic salt; and malt extract, meat extract or the like as other nutrients. The cultivation is carried out in aerobic condition, and generally the cultivation time is about 1 to 5 days, pH of the medium is 3 to 9, and the temperature of cultivation is 10 to 50xc2x0 C.
The reduction process of the present invention is carried out by adding chlorohydroxyacetone derivative as the substrate, coenzyme NAD(P) and culture of the above-mentioned microorganism or treated product thereof to an appropriate solvent, stirring them with adjusting pH.
Though the reaction condition varies in accordance with the enzyme, microorganism or treated product employed and the concentration of the substrate, the concentration of substrate is usually about 0.1 to 90% by weight, preferably about 1 to 50% by weight, the proportion of coenzyme NAD(P) is 0.0001 to 1% by mole, preferably 0.001 to 0.1% by mole based on the substrate, the reaction temperature is 10 to 50xc2x0 C., preferably 20 to 40xc2x0 C. The reaction pH is 4 to 9, preferably 4 to 8. The reaction time is 1 to 120 hours, preferably 4 to 72 hours. The substrate can be added all at once or continuously. The reaction can be carried out batchwise or continuously. In addition, when whole cell is used in the reduction process as enzyme source, the coenzyme may not be added depending on the concentration of the substrate because a certain quantity of coenzyme is present in the whole cell.
Herein, xe2x80x9cculture of microorganismxe2x80x9d means culture solution containing whole cell or cultivated whole cell. xe2x80x9cTreated product thereofxe2x80x9d means, for example, crude extract, lyophilized microorganism, dried microorganism with acetone or ground product of such microorganism by friction. Furthermore, such treated product thereof may be enzyme itself or one obtained by fixing the whole cell as it is by known procedure. The fixation may be carried out in the manner known to a person skilled in the art, for example, cross-link method, physical adsorption method, calthration method.
The amount of costly coenzyme used in the reduction process of the present invention can be significantly reduced by using the common coenzyme NAD(P)H regenerating system jointly. The representative Example of the NAD(P)H regenerating system is the method in which glucose dehydrogenase is used coupled with glucose.
When reaction similar to the above is carried out by using culture of transformed microorganism obtained by introducing, to the host microorganism, a gene coding a reductase and a gene coding an enzyme capable of regenerating coenzyme to which the enzyme depends, or by using a treated product thereof, an optically active chloropronediol derivative can be prepared at a lower cost since it is not necessary to prepare enzyme source necessary for regenerating coenzyme separately.
It is possible to purify the optically active chloropronediol derivative generated by reduction reaction according to general procedure. For example, (S)-1-benzyloxy-3-chloro-2-propanol can be purified by removing suspended materials such as whole cell by means of treatment such as centrifugation or filtration, if necessary, in case of using a microorganism and the like, followed by extraction with an organic solvent such as ethyl acetate or toluene from the mixture, then removing the solvent under reduced pressure, and treating the same by carrying out distillation under reduced pressure or chromatography.
Hereinafter, the present invention is explained in detail by means of Examples, but the present invention is not limited thereto.